SAMT VOL 76 21 OKT 1989 399
The identification of two low-density lipoprotein
receptor gene mutations in South African
familial hypercholesterolaemia
M. J. KOTZE,
E. LANGENHOVEN,
L. WARNICH,
L.
DU PLESSIS,
M. P. MARX,
C. J. J. OOSTHUIZEN,
A.
E. RETIEF
Summary
Two point mutations were discovered in the low-density lipoprotein genes of patients with familial hypercholesterol-aemia (FH). Defective genes were cloned and/or amplified by the polymerase chain reaction (PCR) method and the DNA sequences determined. A guanine to adenine base transition in exon 4 was found to be the molecular defect in 20% of cases of FH in the Afrikaner population. A second mutation, a guanine to adenine base substitution in exon 9, was identified in two homozygous FH individual~. Restriction enzyme ana-lysis of PCR-amplified DNA from blood and tissue samples now permits accurate diagnosis of these mutations.
SAIr Med J1989; 76: 399-401.
Familial hypercholesterolaemia (FH) is an autosomal dominant disease caused by mutations in the low-density lipoprotein receptor (LDL-R) gene located on chromosome 19.1 The
defective receptor causes LDL to accumulate to high levels in plasma, which eventually leads to atherosclerosis and premature heart attacks.
The prevalence of the disease is very high (1 in 80) in the white Mrikaans-speaking section of ·the South Mrican popula-tion.2A founder gene effect has been suggested to explain the high frequency of FH in this so-called Afrikaner population.3
The Afrikaner has indeed a history of a small founder co=u-nity, which remained isolated through religious belief and cultural bonds. We recently reported on the segregation of genetic markers in and around the LDL-R gene in the FH and unaffected Afrikaner populations. Evidence was presented that at least two founders must have been responsible for the high frequency of FH in this population group.4 Haplotype studies showed that a presumed defective gene co-segregated with the rare allele of a Nco I restriction fragment length polymorphism (RFLP)5 in 70% of FH families. A second haplotype, determined by the rare allele of theSruI enzyme,6 co-segregated with a second gene defect in 20% of FH families. This association was confirmed in genetic marker studies in 27 FH homozygotes.7
To date the diagnosis of FH in South Africa relied on the measurement of total or LDL cholesterol levels. It is known
MRC Cytogenetics Research Unit, Department of Human Genetics, University of Stellenbosch, Parowvallei, CP M.
J.
KOTZE, M.Se.E. LANGENHOVEN, M.Se. L.·WAR TICH, M.Se. L. DU PLESSIS, B.se. HO"iS M. P. MARX, M.Se., PH.D.
C.
J. J.
OOSTHUIZEN, Mse., PH.D. A. E. RETIEF, M.Se.,PH.D.Accepred 8 Aug 1989.
Reprint requests [0: Professor A. E. Rerief. Depr of Human Genetics, University of
Steilenbosch Medical School,POBox 63, Tygetberg, 7505 RSA.
-that an elevated level of LDL cholesterol is not always an accurate indication of the disease, since overlapping values are found in affected and unaffected individuals.
We have now cloned the defective genes and by DNA sequence analysis two different single basepair (bp) mutations have been identified. Amplification of genomic DNA from blood and tissue samples by the polymerase chain reaction (PCR)8 method, using synthetic oligonucleotides specific for the mutated exons, now permits accurate diagnosis of these mutations after restriction enzyme digestion and electropho-resis of the DNA.
Material and methods
Patients and families
Blood samples were obtained from the FH patients, their families and normocholesterolaemic individuals, as previously described.4
.7
Genomic cloning
A genomic library was constructed in lambda L47.1 using
Bgl II digested DNA from an FH patient who was known to have the rare allele ofSw I haplotype. A recombinant clone
(FH8-30) was isolated from the library using a 1,05 kbPsr I
subclone of pLDLR-3, a full-length cDNA clone, as a probe.7
Restriction endonuclease mapping and Southern blotting indicated that the 15 kbBgl11clone FH8-30 contained exons 4 -11 of the LDL-R gene. The insert also lacked the Sw I
binding site in exon 8. A 7 kb Hind Ill/Barn HI fragment of the clone was further subcloned into pBR 328 and used for DNA sequencing.
DNA amplification
Genomic and cloned DNA amplification using Taq poly-merase (Amersham) was performed according to the procedure described by Saikier af.9The following oligonucleotide primers (Beckman Instruments) were used for amplification of specific exons of the LDL-R gene:
Exon4: 5' end - 5'-CATCCATCCCTGCAGCCCCC-3' (H) 3' end - 5'-CCATACCGCAGTTTTCCTCG-3' (I) Exon 9: 5' end - 5'-GCTCCATCGCCTACCTCTTC-3'
3' end - 5'-CTGCAGATCATTCTCTGGGA-3' The PCR products were used for either direct DNA sequen-cing or enzyme digestion and electrophoresis.
DNA sequencing
DNA fragments were sequenced by the dideoxy chain-termination methodiOusing the oligonucleotide primers specific for the exons as described above. Both strands were sequenced (Taq Track, Promega). The results were compared with the normal sequence.!I
400 SAMJ VOL 76 21 OCT 1989
Restriction digests of amplified DNA
Aliquots (50 J,LI) of amplified DNA sequences were digested with the specific restriction enzyme and electrophoresed in 2% agarose gels. The DNA fragments were stained with ethidium bromide and visualised by ultraviolet fluorescence.
Results
Single base substitution in exon 4
A single base substirution at position 523 was observed in the DNA sequence of exon 4 of the LDL-R gene cloned from an FH patient in whom the rare allele of theSw I polymor-phism co-segregates with the disease. In Fig. lA the guanine to adenine transition is marked in the partial autoradiogram.
A
This mutation was observed by comparing the cloned sequences with those found in a normal allele. The existence of this mutation was further cOnIlIDled in DNA sequence analysis of 3 additional FH patients who carry the rareSru I allele as a marker.
Restriction enzyme digests
From the DNA sequence data of the normal and mutated exon 4 and the known binding sites of the available restriction enzymes, we deduced that there would be a loss of anMho 11 enzyme binding site in DNA of individuals with the mutated allele. DNA from a normal individual, the cloned fragment (FH8-30) and aSw I-associated FH patient was amplified by peR and digested with Mho 11 (Fig. 2). DNA from the
B
518
G
C
G
A
A
G-.A
A
T
G
G
528
C
EXON4
GAT C
EXON9
GAT C
C
T
G
G
T
A.-G
C
A
A
G
G
1290
1280
Fig. 1. Autoradiograms o.f sequencing gels demonstrating point mutations in the LDL-R gene of Afrikaner FH patients. A. The mu.tantse9~enceInexon 4 of aStuI-associated allele. A guanine to adenfollikeline transition (G - A) is indicated
at nucleotide POSition 523. B. The mutant sequence of an Nco I-associated FH homozygote. A single base change from guanine to adenine is indicated at nucleotide position 1285.
EXON 4
H
H
~~
...
H
I
bp
1
2
3
400
220
180
Fig. 2. Gel electrophoresis ofMboII-digested peR-amplified exon 4 DNA. The diagram shows the mutation in exon 4 and the expected fragment sizes after Mbo11 digestion. Lane 1: DNA from a normal control individual. Lane 2: A cloned fragment from the rareStuI allele. Lane 3: DNA from a
normal individual was digested to completion, and fragments of 220 and 180 bp were observed. The digestion of DNA from the cloned fragment resulted in one fragment of 400 bp indicating loss of theMbo II binding site (lane 2). DNA from a FH heterozygote showed heterozygosity after digestion by
Mbo II (lane 3). This confirms that the enzyme did not digest the DNA of the mutated allele, while that of the normal allele was digested.
Analysis of the exon 4 mutation in normal
and FH families
The presence of the exon 4 mutation, using PCR-amplified DNA and restriction digestion withMbo II, was studied in 2 informative FH families of whom 10 members were FH hetero-zygotes with the associated rare Sru I allele. The presence of
the mutation, as seen by the absence of theMbo II restriction site in the amplified DNA of affected heterozygotes, correlates with high LDL cholesterol levels in these individuals. This is a further confirmation that the exon 4 mutation dQes indeed cause the disease in these families.
The absence of the mutation in 20 normal individuals, of whom 6 were also found to have the associated rare Sru I
allele, further substantiates this finding.
Single base substitution in exon 9
A single base substitution at position 1285 was observed in the DNA sequence of exon 9 from amplified DNA of 2 FH homozygotes, in whom the rare allele of theNco I RFLP co-segregates with the disease. In Fig. IB the guanine to adenine transition is marked in the partial autoradiogram. The mutation was observed by comparing the sequence data of exon 9 of both alleles of two homozygotes with the sequence data from normal controls.
From the sequence data of both the normal and the mutated allele of exon 9, we deduced that there would be a loss of the restriction enzyme binding site of the enzyme Mae II in individuals with the mutated allele. Using the oligonucleotides for exon 9 as primers, the amplified DNA from true homo-zygotes would not digest using the enzyme Mae II. The heterozygotes would show a similar heterozygosity in fragment sizes as was illustrated for exon 4 mutation. The Mae II enzyme was not readily available for further studies prior to
publication.
-SAMT VOL. 76 21 OKT 1989 401
Discussion
DNA" from a compound homozygote was cloned and the Sw
I-associated allele of the LDL-R gene was isolated from the library for further characterisation. A point mutation was observed in exon 4 of this allele, causing loss of an Mho II binding site. Evidence that the mutation causes the disease in these families was demonstrated in studies where the mutation was seentosegregate with high LDL cholesterol levels. Preli-minary data indicate that this mutation occurs in all Sru
1-associated FH patients. Extrapolation of the haplotype data indicates that the exon 4 mutation will account for about 20% of the founder-related defect in the Afrikaner population.
In 2 FH homozygotes with the associated Nco I allele a point mutation was found in exon 9. This mutation resulted in the loss of anMaeII binding site. Population screening for the incidence of this mutation in FH patients will reveal whether all the Nco I-associated FH patients have the same exon 9 mutation of the gene.
Accurate diagnosis of these mutations is now possible by
Mho II andMae II digestion of PCR-amplified DNA.
This study has shown that direct molecular diagnosis of these gene mutations in familial hypercholesterolaemia is simple, reliable and allows for rapid unequivocal diagnosis of the disease. Identification of the defective genes in hetero-zygous parents also permits the prenatal diagnosis of homo-zygosity in chorionic or amniotic tissue of the fetus and offers the possibility for termination of the pregnancy.
REFERENCES
I. Russell DW, Esser V, Hobbs HH. Molecular basis of familial hypercholes-tetolemia.Arteriosclerosis1989; 9: supplI,1-8 - 1-13.
2. Jooste PL, Benade AJS, Rossouw JE. Ptevalence of familial hypetcholestero-laemia in three rural South African communities.SAfr MedJ 1986; 69: 548-551.
3. Seftel HC, Baker SG, Sandlet MPec al.A host of hypercholesterolaemia homozygotes in South Africa.Br MedJ 1980; 281: 633-636. . .
4. Kotze MJ, Langenhoven E, Retief AE eCal.Haplotype aSSOCiations of three DNA polymotphisms at the human low denslry lipoprotem gene locus m familial hypercholesterolaemia.J Med Genec1987; 24: 750-755.. . 5. Korze MJ, Langenhoven E, Dietzsch E, Retief AE. A RFLP assopated with
the low densiry lipoprotein teceptor gene.Nucleze ACIds Res1987; 15: 376. 6. Kotze MJ, Retief AE, Brink PA, Weich HFH. A DNA polymorphism m the
human low densiry lipoprotein receptor gene.SAfr MedJ1986; 70: 77-79. 7. Korze MJ, Langenhoven E, Retief AEec al. Haplorypes Identified byIQ
DNA restriction fragment length polymorphlsms at the human low denSity lipoprotein receptor gene locus.JMed Genec1989; 26: 255-259. . 8. Mullis KB, Faloona FA. Specific syntheSIS of DNA m vitro via a
poly-merase-catalyzed chain reaction.Mechods Enzymol1987;155: 335-349. . 9. Saiki RK, Gelfand DH, Stoffel Sec al.Pnmer-dltected enzymatlc
amplIfica-tion of DNA with a thermostable DNA polymerase. Science 1988; 239: 487-491. . . . . 10. Sanger F, Nicklen S, Coulson AR. DNA sequencing With cham-termmatmg
inhibitors.Proe Nacl AeadSeiUSA1977; 74: 5463-5467.
11. Yamamoto T Davis GG, Brown MS eC al. The human LDL receptor: a cysteine-rich protein with multiple Alu sequences in its mRNA.Cell 1984; 39: 27-38.